Hybrid seed production

ABSTRACT

Methods of preparing hybrid seed are described. One such method comprises interplanting a male parent plant which is male fertile and homozygous recessive female sterile and a female parent plant which is homozygous recessive male sterile and female fertile, allowing cross-pollination and obtaining the seed produced therefrom. The genomic material of each parent plant may also have integrated therein a gene construct comprising a promoter sequence-responsive to the presence or absence of an exogenous chemical inducer, optionally operably linked to one or more enhancer or intron sequences, operably linked to a gene which fully restores the fertility of each parent plant, the gene being expressed by the application to the plant of an external chemical inducer thereby allowing each parent to self-pollinate.

This application is a 371 of PCT/GB99/00238 filed Jan. 22, 1998.

FIELD OF THE INVENTION

The present invention relates to methods of preparing hybrid seed.

In particular, the present invention relates to the molecular control ofsterility in crop plants. Such male and female sterility in plants canbe used in the preparation of hybrid seed from crops which are naturallyself-pollinators.

The present invention also provides for a method of restoring fertilityin the parent plants to allow self-pollination, thereby allowing themaintenance of the parental lines.

The present invention further relates to expression cassettes forincorporation into plants and to the use of such expression cassettes ina male/female sterility restorer system.

Hybrid plants grown from hybrid seed benefit from the heterotic effectsof crossing two distinct genetic backgrounds. The production of hybridseed depends on the ability to control self-pollination and ensurecross-pollination of male and female parent plants.

A number of methods are available to control pollen fertility. Forexample, in the case of maize, which has separate male and femaleflowers, control of pollen fertility is achieved by physically removingthe male inflorescence or tassel, prior to pollen shed, thus preventingself-pollination.

Most major crops, however, have both functional male and femalereproductive organs within the same flower. In this instance, removal ofthe pollen producing organs is very labour intensive and expensive. Theuse of chemicals (gametocides), particularly in wheat, maize (corn) andrice, to kill or block pollen production produces transitory malesterility but the use of such chemicals is expensive. The reliability ofthe chemicals and their length of action are also issues.

There is considerable interest in developing systems of pollen controlbased on genetic mechanisms producing male sterility. There are twogeneral types: a) nuclear male sterility caused by the failure of pollenproduction due to one or more nuclear genes and b) cytoplasmic malesterility (CMS) in which pollen production is blocked because of adefect in a gene in the mitochondria.

Currently available nuclear systems are based on the introduction of amale sterility trait to one parent plant followed by the introduction ofa fertility restoration gene as a result of cross-pollination withanother plant to produce fertile hybrid plants. The Paladin system,which is described in WO96/01799, is different and is based on theseparation during hybrid seed production of genes which, when expressedtogether in one plant, have a cytotoxic effect leading to malesterility.

Rice and wheat are self-pollinating plants and have small hermaphroditeflowers and so the detasseling approach taken for hybrid seed productionin maize is not applicable. Manual removal of anthers is difficult andtime consuming. Moreover, wheat pollen is relatively heavy and is viableonly for a short time, rarely remaining viable for longer than 30minutes. The technique of planting used in hybrid corn production i.e.planting the male parent in a block physically separated from the femaleparent (the male sterile) and allowing wind pollination does not,therefore, work well in wheat or rice. The male and female parents forthese crops have to be interplanted to ensure cross pollination. Ashybrid seed needs to comprise more than 95% hybrids, it is necessary toremove seed arising from self-pollination of the male parent or to makethe male parent incapable of self-fertilisation and therefore incapableof producing non-hybrid seed. Clearly, the interplanting of the parentplants means that the first option is difficult unless the male plantsare susceptible to some chemical treatment to which the female parent istolerant e.g. herbicide treatment.

Our International Patent Application No. PCT/GB90/00110 describes acascade of gene sequences which expresses a protein which disrupts thebiosynthesis of viable pollen in a female parent plant. In this case,however, only one of the parent plants i.e. the female parent is sterileto minimise self-pollination of the female plant and this female plantis crossed with a fertile male parent plant to yield fertile hybridseed. There is no description in the literature, however, of a method ofproducing hybrid seed wherein both parent plants are unable toself-pollinate.

SUMMARY OF THE INVENTION

The present invention relates to two methods by which hybrid seed may beproduced which seeks to overcome the problems presently associated withthe production of hybrid seed, particularly with the production ofhybrid wheat and rice seed.

According to a first aspect of the present invention, there is provideda method of preparing hybrid seed comprising interplanting a male parentplant which is male fertile and homozygous recessive female sterile anda female parent plant which is homozygous recessive male sterile andfemale fertile, allowing cross-pollination and obtaining seed producedtherefrom.

According to a second aspect of the present invention, there is providedthe use of the above method to produce hybrid seed.

According to a third aspect of the present invention, there is providedfertile plants produced by the aforementioned method.

According to a fourth aspect of the present invention there is providedthe progeny of the aforementioned plants, the seeds of such plants andsuch progeny.

According to a fifth aspect of the present invention there is providedan expression cassette comprising:

(a) a first gene promoter sequence which is a male flower specificpromoter sequence;

(b) a disrupter gene encoding a product capable of disrupting malefertility operably linked to the first gene promoter sequence;

(c) a second gene promoter sequence which is a female flower specificpromoter sequence optionally operably linked to one or moretranslational enhancer or intron sequences;

(d) a restorer gene encoding a product capable of restoring femalefertility operably linked to the second gene promoter sequence;

(e) a third gene promoter sequence responsive to the presence of anexogenous chemical inducer optionally operably linked to one or moretranslational enhancer or intron sequences; and

(f) a restorer gene encoding a product capable of restoring malefertility operably linked to the third gene promoter sequence;

whereby the presence of the exogenous chemical inducer controls malefertility.

According to a sixth aspect of the present invention there is providedan expression cassette comprising:

(a) a first gene promoter sequence which is a female flower specificpromoter sequence;

(b) a disrupter gene encoding a product capable of disrupting femalefertility;

(c) a second gene promoter sequence which is a male flower specificpromoter sequence optionally operably linked to one or moretranslational enhancer or intron sequences;

(d) a restorer gene encoding a product capable of restoring malefertility operably linked to the second gene promoter sequence;

(e) a third gene promoter sequence responsive to the presence or absenceof an exogenous chemical inducer optionally operably linked to one ormore translational enhancer or intron sequences; and

(f) a restorer gene encoding a product capable of restoring femalesterility operably linked to the third gene promoter sequence;

whereby the presence of the exogenous chemical inducer controls femalefertility.

According to a seventh aspect of the present invention there is provideda further method of producing hybrid seed comprising incorporating afirst expression system according to the fifth aspect of the presentinvention into a first plant to generate a hemizygous female parentplant and incorporating a second expression system according to thesixth aspect of the present invention into a second plant to generate ahemizygous male parent plant;

applying an exogenous chemical inducer to the transformants therebyallowing the plants to self-pollinate;

growing up plants from the resulting seed;

selecting for male and female homozygous plants;

crossing the selected male and female plants; and

obtaining the resulting hybrid seed.

According to an eighth aspect of the present invention there is providedplant tissue transformed with either one of the expression cassettes asdefined above and material derived from the said transformed planttissue.

According to a ninth aspect of the present invention there is providedfertile whole plants comprising the tissue or material as defined above.

According to a tenth aspect of the present invention there is providedthe progeny of the selected plants produced according to the seventhaspect of the present invention, the progeny comprising expressioncassettes as defined above incorporated, preferably stably incorporated,into their genome and the seeds of such plants and such progeny.

According to an eleventh aspect of the present invention, there isprovided a plant, the genome of which comprises the first expressioncassette according to the fifth aspect of the present invention.

According to a twelfth aspect of the present invention, there isprovided a plant, the genome of which comprises the second expressioncassette according to the sixth aspect of the present invention.

According to a thirteenth aspect of the present invention, there isprovided hybrid seed produced by crossing these two plants and obtainingthe resulting hybrid seed produced therefrom.

According to an fourteenth aspect of the present invention there isprovided the use of the second method according to the present inventionto produce hybrid seed.

According to a fifteenth aspect of the present invention there isprovided a method of transforming a plant comprising incorporating intothe genome of the plant an expression cassette as defined above whereinthe restorer gene, which is operably linked to a third gene promotersequence, is inducibly expressed in the target tissue but may beconstitutively expressed in one or more other tissues so that thedisrupter gene is only effective in the target tissue. The thirdpromoter sequence may be constitutively expressed at a particular stagee.g. in callus tissue.

Preferably, the first method of the present invention wherein thegenomic DNA of each parent plant has integrated therein a gene constructcomprising a promoter sequence responsive to the presence or absence ofan exogenous chemical inducer, optionally operably linked to one or moretranslational enhancer or intron sequences, operably linked to a genewhich fully restores the fertility of each parent plant, the gene beingexpressed by the application to the plant of an external chemicalinducer thereby allowing each parent to self-pollinate.

Preferably, the female parent plant is homozygous for a recessive genewhich disrupts the biogenesis of viable pollen or which significantlyreduces the viability of the pollen.

Preferably, the male parent plant is homozygous for a recessive genewhich disrupts female floral structures such as ovule, style, stigma insuch a way that fertilisation is prevented, or adhesion, hydration orgermination of pollen inhibited or which inhibits pollen tube growth orguidance.

Preferably, the inducible promoter sequence is the AlcA promotersequence or the GST-27 promoter sequence.

Preferably, the parent plants are wheat, barley, rice, maize, sugarbeet,tomato, sunflower, canola, cotton, soybean and other vegetables such aslettuce.

Preferably, the F1 hybrid seed produced by the first method of thepresent invention gives rise to plants, all of which are fully fertile.

Preferably, the F2 hybrid seed produced by the first method of thepresent invention gives rise to plants which segregate for sterility,about 25% being female sterile.

Preferably, the sterility of the parents is caused by a natural orgenetically manipulated mutation.

Preferably, the first expression cassette defined above and used in thesecond method of the present invention comprises a disrupter geneencoding a product which is capable of disrupting pollen production.

Preferably, the first expression cassette defined above comprises adisrupter gene encoding a product which is capable of being expressed inthe tapetal cells of the plant.

Preferably, the third gene promoter sequence in the first expressioncassette is the AlcA promoter sequence or the GST-27 promoter sequence.

Preferably, the second expression cassette defined above and used in thesecond method of the present invention comprises a restorer geneencoding a product which is capable of restoring pollen production.

Preferably, the second expression cassette defined above comprises arestorer gene which is capable of overcoming disruption of the tapetalcells.

Preferably, the third gene promoter sequence in the second expressioncassette is the AlcA promoter sequence or the GST-27 promoter sequence.

Preferably, the male plants in the second method according to thepresent invention comprise a homozygous dominant gene restoring malefertility.

Preferably, the female plants in the second method according to thepresent invention comprise a homozygous dominant gene restoring femalefertility.

Preferably, the F1 hybrid seed produced gives rise to plants, theanthers of which produce approximately 50% of viable pollen, where thefirst gene promoter sequence of the first expression cassette is agametophytic promoter sequence.

Preferably, the F1 hybrid seed produced gives rise to plants, all ofwhich are fully fertile where the first promoter sequence of the firstexpression cassette is a sporophytic promoter sequence.

Preferably, the F2 hybrid seed gives rise to plants which segregate forsterility, of which a significant number are female sterile.

Preferably, the male and female homozygous plants produced by the secondmethod according to the present invention are multiplied and maintainedby the application of an exogenous chemical inducer to the plants,thereby allowing the plants to self-pollinate. In this regard, furthergenerations of self-pollination of the selected male and femalehomozygous plants can be produced and when hybrid seed is required, theplants may be crossed to obtain hybrid seed.

Preferably, the plants used in the second method of the presentinvention are wheat, barley, rice, maize, sugarbeet, tomato, sunflower,canola, cotton, soybean and other vegetables.

Preferably, the restorer gene used in the method of transforming a plantaccording to the present invention is constitutively expressed in callustissue from which transformed plants are regenerated.

Preferably, the restorer gene is inducibly expressed in the male orfemale flower structures.

Preferably, the third gene promoter sequence of the expression cassettesused in the transformation process is the GST-27 or the AlcA promotersequence.

A preferred embodiment of the present invention is a method of preparinghybrid seed comprising interplanting a male parent plant which is malefertile and homozygous recessive female sterile and a female parentplant which is homozygous recessive male sterile and female fertile,allowing cross-pollination and obtaining seed produced therefrom whereinthe genomic DNA of each parent plant has integrated therein a geneconstruct comprising a promoter sequence responsive to the presence orabsence of an exogenous chemical inducer operably linked to a gene whichfully restores the fertility of each parent plant, the gene beingexpressed by the application to the plant of an external chemicalinducer thereby allowing each parent to self-pollinate when required formultiplication of the seed stocks of each parent plant.

A further preferred embodiment of the present invention is an expressionsystem comprising:

(a) a first gene promoter sequence which is a male flower specificpromoter sequence;

(b) a disrupter gene encoding a product capable of disrupting malefertility operably linked to the first gene promoter sequence;

(c) a second gene promoter sequence which is a female tissue specificpromoter sequence optionally operably linked to one or moretranslational enhancer or intron sequences;

(d) a restorer gene encoding a product capable of restoring femalefertility operably linked to the second gene promoter sequence;

(e) a third gene promoter sequence responsive to the presence or absenceof an exogenous chemical inducer optionally operably linked to one ormore translational enhancer or intron sequences;

f) a restorer gene encoding a product capable of restoring malefertility operably linked to the third gene promoter sequence;

whereby the presence of the exogenous chemical inducer controls malefertility, wherein the gene capable of disrupting male sterility is adisrupter gene encoding a product which is expressed in the tapetalcells of the plant.

Another preferred embodiment of the present invention is an expressionsystem comprising:

(a) a first gene promoter sequence which is a female tissue specificpromoter sequence;

(b) a disrupter gene encoding a product capable of disrupting femalefertility;

(c) a second gene promoter sequence which is a male tissue specificpromoter sequence optionally operably linked to one or moretranslational enhancer or intron sequences;

(d) a restorer gene encoding a product capable of restoring malefertility operably linked to the second gene promoter sequence;

(e) a third gene promoter sequence responsive to the presence or absenceof an exogenous chemical inducer optionally linked to one or moretranslational enhancer or intron sequences; and

(f) a restorer gene encoding a product capable of restoring femalefertility operably linked to the third gene promoter sequence;

whereby the presence of the exogenous chemical inducer controls femalefertility and wherein the gene capable of restoring male fertility is agene which encodes a product which restores pollen production in thetapetal cells.

The preferred male flower specific promoter sequences are the maizeMSF14 and C5 (derived from pectin methyl esterase) promoter sequences.

The term “plant material” includes a developing caryopsis, a germinatingcaryopsis or grain, or a seedling, a plantlet or plant, or tissues orcells thereof, such as the cells of a developing caryopsis or thetissues of a germinating seedling or developing grain or plant (eg inthe roots, leaves and stem).

The term “cassette” which is synonymous with terms such as “construct”,“hybrid” and “conjugate” includes a gene of interest directly orindirectly attached to a gene promoter sequence. An example of anindirect attachment is the provision of a suitable spacer group such asan intron or enhancer sequence intermediate the promoter and the gene ofinterest. Such constructs also include plasmids and phage which aresuitable for transforming a cell of interest.

The term “disrupter gene” is a gene which acts in a dominant fashion,and when expressed at a suitable stage of plant development, will leadto the failure of a plant to form normally functioning female flowerstructures or normally functioning male flower structures so that theplant is female or male sterile. Such a gene may exert its effect bydisrupting tissues such as the tapetum and endothelium. The gene may beexpressed specifically in male flowers during pollen formation causingcell death of the anthers and associated tissues, pollen mother cells,pollen and associated tissues. It may also be expressed in the stigma orin the transmitting tract of the style, thus interfering with theprocess of pollen adhesion, hydration, pollen germination and pollentube growth and guidance. The origin of the disrupter genes can be froma variety of naturally occurring sources e.g. human cells, bacterialcells, yeast cells, plant cells, fungal cells, or they can be totallysynthetic genes which may be composed of DNA sequences, some of whichmay be found in nature, some of which are not normally found in natureor a mixture of both. These genes will preferably have an effect onmitochondrial metabolism, as it is known that a good energy supply is anabsolute requirement for the production of fertile pollen. The disruptergenes may, however, be effectively targeted to other essentialbiochemical functions such as DNA and RNA metabolism, protein synthesis,and other metabolic pathways. The preferred dominant disrupter gene isbarnase.

The term “restorer gene” is a gene which acts in a dominant fashion, andwhen expressed, will reverse the effects of the disrupter gene. Thepreferred dominant restorer gene is barstar.

The term “female flower” is intended to include all parts of the femalereproductive organs including but not limited to, ovary, ova, pistil,style, stigma, transmitting tract, placenta.

The term “male flower” is intended to include all parts of the maleflower, including but not limited to, the tapetum, anthers, stamens,pollen.

The methods of hybrid seed production according to the present inventionare different from and have a number of advantages over existing methodsin a number of ways. The utilisation of both male and female sterilityhas not previously been described. This feature prevents selfpollination of either parent thus allowing the production of hybrid seedwithout the need for separate planting blocks for male and femaleparents. This interplanting of male and female parent plants maximisesthe opportunity for cross pollination in crops, such as wheat and rice,which are essentially self pollinators. In the examples of wheat andrice, where block planting is not carried out, this method allowsproduction of hybrid seed without the need to apply herbicide to rogueout male parent plants after fertilisation of the female parent. Achemically inducible restorer system is needed only for the maintenanceof homozygous parental lines rather than for the hybrid seed productionprocess. This means that chemicals are applied to limited acreages andthen only infrequently. A number of disrupter-restorer systems, oroperator-repressor systems may be used in the present invention.

Plants containing the expression cassettes of the present inventionwhich control male and female fertility may also be used separately tomake F1 hybrids with other parent lines, which do not contain theexpression cassettes, if suitable alternative control of male or femalefertility (such as mechanical removal of anthers or ovules, or use ofchemical gametocides) is used in the other line. If the progeny fromthese F1 hybrids are then backcrossed for an appropriate number ofgenerations to the other hybrid parents, whilst selecting for thepresence of the expression cassette with molecular, biochemical orprogeny-testing techniques, the system for controlling male or femalefertility can be transferred or introgressed into new parentbackgrounds. Alternatively, F1 hybrids with other parent lines can beself-pollinated, through application of an exogenous chemical inducer torestore male or female fertility as appropriate, so as to select newhybrid parents containing the expression cassettes, through the normalprocess of plant breeding. Use of such introgression and plant breedingwill permit the methods of hybrid seed production of the presentinvention to be used with a wide variety of new and existing F1 hybridparental combinations.

Promoters which are inducible by application of exogenous chemicals areknown in the art. Suitable inducible promoters are those which areactivated by application of a chemical, such as a herbicide safener.Examples of inducible promoters include AlcA/R switch system describedin our International Publication No. WO. 93/21334, the GST switch systemdescribed in described in International Publication Nos WO 90/08826 andWO 93/031294 or the ecdysone switch described in InternationalPublication No. WO 96/37609. Such promoter systems are herein referredto as “switch promoters”. The switch chemicals used in conjunction withthe switch promoters are agriculturally acceptable chemicals makingthese promoters particularly useful in the methods of the presentinvention.

One of the advantages of using the AlcA promoter, which is a componentof the Alc A/R switch system, in the present invention is that thechemical inducer used is ethanol. This chemical is advantageous in thatit can be applied as a root drench, as an aqueous spray, or as a gas. Itis effective at concentrations of 1% and is non-toxic to operators andto the environment.

The present invention can be used for any mono- or di-cotyledonous plantwhich the breeder or grower wants to produce as F1 hybrid seed and forwhich suitable transformation techniques are or become available,particularly wheat and rice crops. The present invention has theadvantage of reducing crop management costs associated with the F1hybrid seed production, ease of purity control of hybrid seed andmaintenance of parental lines.

In a particular application, the present invention relates to theproduction of male and female parental plants, which are renderedsterile using molecular engineering techniques. The sterility of theseplants can be reversed by using a chemical application which leads tothe restoration of fertility.

The anther is the site of male reproductive processes in floweringplants. It is composed of several tissues and cell types and isresponsible for producing pollen grains that contain the sperm cells.The tapetum is a specialised tissue which plays a critical role inpollen formation. It surrounds the pollen sac early in pollendevelopment, degenerates during the latter stages of development and isnot present in an organised form in the mature anther. The tapetumproduces a number of compounds which aid pollen development or areincorporated into the pollen outer wall and it has been demonstratedthat many of the natural male sterility mutations have impaired tapetumdifferentiation or function. Tapetal tissue is therefore critical to theformation of functional pollen grains.

A number of genes have been identified and cloned that are specificallyexpressed in tapetal tissue. They include Osg6B, Osg4B (Tsuchiya et al.1994, Yokoi, S et al. 1997), pE1, p T72 (WO9213957), p CA55 corn(WO92/13956), TA29, TA13, (Seurinck et al 1990), RST2 corn (W09713401),MS14,18,10 and A6, A9 from Brassica napus (Hird et al. 1993).

A tapetum specific promoter isolated from rice has been shown to giverise to male sterile plants when used to drive expression of β 1,3glucanase in tobacco, (Tsuchiya et al. 5 1995). The tapetum specificpromoter TA29 has been used to produce male sterile tobacco (Mariani etal 1990) and pCA55, pE1 and pT72 to produce male sterile wheat (De Blocket al. 1997) when driving the expression of barnase.

Pollen specific clones have been obtained from a number of species,including corn (Hanson et al. 1989, Hamilton et al. 1989,) and tomato(Twell et al. 1990, 1991).

Anther specific clones have been isolated from a number of species Bp4Aand C from Brassica napus (Albani et al. 1990), chs from petunia (Koeset al. 1989), rice (Xu et al. 1993, Zou et al. 1994), amongst others.

Wheat homologues of these clones and others may be obtained by suchmethods as degenerate PCR, utilising sequences found in the literature,and subsequent screening of wheat and other genomic libraries, andanalysis of tissue specificity using the expression of reporter genes.These methods are well documented in the literature.

In higher plants the female reproductive organ is represented by thepistil, composed of the ovary, style and stigma. The gynoecium has beenshown to contain up to 10,000 different mRNAs not present in otherorgans (Kamalay and Goldberg 1980). These include regulatory genesresponsible for controlling pistil development as well as “downstream”ones encoding proteins associated with differentiated cell types in thepistil. Genes governing self-incompatibility and their homologues areone class of gene with pistil predominant expression patterns (Nasrallahet al. 1993). Other cloned genes include β glucanase (Ori et al. 1990),pectate lyase (Budelier et al. 1990) and chitinase (Lotan et al. 1989)which are expressed in the transmitting tissue and a proteinaseinhibitor (Atkinson et al. 1993) which are expressed in the style.Others are pathogenesis related or are homologues of genes involved inthe cleavage of glycosidic bonds. These enzymes may facilitate pollentube growth by digesting proteins in the tissue through which the pollentube grows.

A number of female sterile mutants have been identified in Arabidopsis.For example, sin1 (short integument) (Robinson-Beers et al. 1992) andbel1 (bell) (Robinson-Beers et al. 1992) affect ovule development. TheAintegumenta mutation blocks megasporogenesis at the tetrad stage(Elliot, R. C, et al. 1996, Klucher, K. M, 1996). A lethal ovule 2mutation has been observed but not cloned in maize (Nelson et al. 1952).Pistil specific basic endochitinases have been cloned from a number ofspecies (Ficker et al. 1997, Dzelzkalns et al. 1993, Harikrishna et al.1996, Wemmer et al. 1994) and extensin-like genes have been shown to beexpressed in the styles of Nicotiana alata (Chen C-G, et al. 1992).

The following are ovule specific clones ZmOV23,13, (Greco R., et al.unpublished), OsOsMAB3A (Kang H. G., et al. 1995), ZmZmM2 (Theissen G.,et al. 1995) and stigma specific stig1 (Goldman, M. H et al. 1994),STG08, STG4B12 (EP-412006-A). Goldman et al. used the promoter from theSTIG1 gene to drive expression of barnase in the stigmatic secretoryzone. This led to flowers having no secretory zone in the pistils andthus were female sterile. Pollen grains were able to germinate but wereunable to penetrate the surface.

Seven ovule specific cDNAs have been isolated from orchid (Nadeau et al.1996). Again, wheat homologues of these and any others may be obtainedby standard molecular biology techniques.

Another aspect to the methods of the present invention is theidentification of genes impacting on male and female sterility. Suchgenes can be used in a variety of systems to control fertility.

The procedure for tagging maize genes with transposable elements hasbeen reviewed (Doring, 1989). One of the methods which can be used is tocross a maize line carrying active transposable elements and a dominantallele of the target gene with a normal maize strain that does not carrytransposable elements. Progeny from the cross can be selfed and screenedfor the most desirable mutations, i.e. those that lead to sterility. Thesterile plants represent potential instances in which a transposableelement has transposed to a locus bearing a gene essential forfertility. The genes may then be recovered in a variety of ways. U.S.Pat. No. 5,478,369 describes the isolation by this method of a genedescribed as MS45.

A male fertility gene has been identified in Arabidopsis thaliana usingthe En/Spm-I/dSpm transposon tagging system to obtain a male sterility 2(ms2) mutant and the MS2 gene (Aarts et al. 1993). This MS2 gene hasbeen shown to be involved in male gametogenesis, cell wall synthesisdoes not proceed after microspore mother cell meiosis and themicrospores are eventually degraded. Homologues of MS2 have beenidentified in Brassica napus, Zea Mays and to an open reading framefound in wheat mitochondrial DNA. The isolation of genes critical tofertility in Arabidopsis may therefore lead to the cloning of homologuesin other species. This approach can clearly be taken to isolate othergenes critical to fertility.

There is now evidence for the existence of extensive regions ofconserved colinearity among grass species at the genetic level. Ahn andTanksley (1993) showed the relationship between rice and maize andKurata et al (1994) showed that the wheat genome could be aligned withrice and Moore et al (1995) showed that all three maps could be aligned.This opens the way to the use of comparative genome mapping as a meansof gene isolation.

The microsynteny approach to gene cloning is based on the emergingsimilarity in molecular marker and gene order among evolutionary relatedspecies. This approach is particularly attractive for large genomecereal species of agricultural importance like wheat, maize and barleythat may take advantage of their small genome relative, rice. Kilian etal. (1997) report on progress on map-based cloning of the barley RpgIand rpg4 genes using rice as an intergenomic mapping vehicle.

As the above approach is limited to target genes which have beengenetically mapped, an alternative method of gene isolation, which is aneffective transposon tagging system, is being developed in rice usingthe maize Ac/Ds system, Izawa et al (1997).

A number of methods have been suggested as being useful to inactivategenes necessary for fertility or to produce cytotoxic compounds in thetissues to prevent normal development of gametophytes.

Our International patent application no. PCT/GB96/01675 describes amethod of inhibiting gene expression in a target plant tissue using adisrupter gene selected from zANT, tubulins, T-urf, ATP-ase subunits,cdc25, ROA, MOT.

There are several other known inactivating systems. For example, barnase(Mariani et al. 1990), diphtheria toxin A-chain, pectate lyase. Twoexamples of expressing cytotoxic compounds previously described areavidin expression and IamH/IamS,

The expression of β-1,3-glucanase in tapetal cells has been shown togenerate male sterile plants (Worrall et al. 1992). Anti-sense has beenproposed as a mechanism by which the expression of genes critical topollen development can be down regulated and it has been shown (Van derMeer 1992) that antisense inhibition of flavonoid biosynthesis doesindeed lead to male sterility. Reduction of flavanol expression has beenclaimed in maize to result in male sterility (WO 93 18171 PioneerHi-Bred International). Other mechanisms have also been described (Spenaet al. 1992).

Baulcombe (1997) describes a method of gene silencing in transgenicplants via the use of replicable viral RNA vectors (Amplicons™) whichmay also be useful as a means of knocking out expression of endogenousgenes. This method has the advantage that it produces a dominantmutation i.e. is scorable in the heterozygous state and knocks out allcopies of a targeted gene and may also knock out isoforms. This is aclear advantage in wheat which is hexaploid. Fertility could then berestored by using an inducible promoter to drive the expression of afunctional copy of the knocked out gene.

Kempin et al. (1997) report the targeted disruption of a functional geneusing homologous recombination. This method normally, however, producesa recessive mutation i.e. is scorable only in the homozygote. To bedetectable in the heterozygous state it would either have to be lethaldirectly or, for example, cause a block in a pathway such that there wasa build up of a cytotoxic compound leading to lethality. In order todetect a sterility mutation it would be necessary to generate arecessive homozygote by self-pollination, where one in four of theprogeny would be sterile. The switch construct allowing expression ofthe knocked out gene would have to be introduced into a heterozygoteobtained by back crossing the homozygote. This is a potentially usefulmethod for generating the recessive mutants needed for the method laterdescribed in Example 1.

Ribozymes are RNA molecules capable of catalysing endonucleoolyticcleavage reactions. They can catalyse reactions in trans and can betargeted to different sequences and therefore are potential alternativesto antisense as a means of modulating gene expression. (Hasselhof andGerlach). Wegener et al (1994) have demonstrated the generation of atrans-dominant mutation by expression of a ribozyme gene in plants.

Several methods are known for altering plant self incompatibilty systemsby modifying S-gene expression as a means of introducing male sterilityin which a plant is transformed with a construct utilising agametophytic S-gene encoding a ribonuclease in such a way that aself-incompatible plant is converted to self compatible or that aself-compatible plant is converted to self-incompatibility, thuspreventing self pollination.

Examples of combinations of disrupter/restorer genes include barnase andbarstar, and TPP and TPS. The use of the barnase/barstar system tofirstly generate sterility and then restore fertility has been described(Mariani et al. (1992). Trehalose phosphate phosphatase (TPP) whenexpressed in tapetal cells of tobacco using the tapetum specificpromoter Tap1 (Nacken et al 1991) from Antirrhinum results in malesterility. It is thought to be as a result of changing the carbohydratemetabolic and photosynthetic capacity of the tissues in which it isexpressed. Anthers show signs of necrosis and any pollen produced isdead. Back crossing with wild type tobacco results in normal seeddevelopment. Analysis of the progeny shows that sterility segregateswith the transgene. TreC, trehalose-6-phosphate hydrolase is a secondgene whose expression perturbs of levels of trehalose-6-phosphate and ithas been shown that when it is expressed using the constitutiveplastocyanin promoter the result is bud excision before flowering. Thus,if expression is limited to the tapetum, male sterility may result inthe same way as when TPP is expressed in the tapetum. It has also beenshown that after GA application flower buds remain on the plant and somepollen is produced leading in some cases to seed production.

It has also been shown that simultaneous equimolar expression oftrehalose phosphate synthase (TPS) and TPP gives no effect on plantphysiology i.e. TPS counteracts the effect of TPP on carbohydratemetabolic and photosynthetic capacity of tissues in which they areexpressed. It has also been shown that it is possible to restorefertility by retransforming sterile tobacco lines with a constructexpressing TPS in the tapetun. Clearly, expression of TPS could also beput under control of an inducible promoter to allow fertility to berestored when desired, or optimising GA application could be analternative means of restoring fertility. The promoter from a geneexpressed specifically in the tissues surrounding or in, the ovule, suchas the MADS box gene FBP7 could be used to drive expression of TPP orTreC to obtain female sterility. It is likely that some optimisation ofcodon usage may be required to obtain the same effect in a monocot cropplant such as wheat or corn, (Merlo and Folkerts), (Seed and Haas).

The use of a number of operator/repressor systems has been described asa means of controlling gene expression in plants. Wilde et al.demonstrated the use of the E. coli lac system to repress expression ofGUS under the control of the maize cab promoter (lac I expression drivenby 35S CaMV promoter). Operator sequences were inserted at variouspositions within the CAB promoter and the extent of repression assessed.Depending upon the position of the operator sequences, a range ofrepression was observed. When the operator sequence was incorporated byreplacement between the TATA box and the transcription start, repressionof ˜90% was obtained. This repression can be relieved by the addition ofIPTG. This was shown both in tobacco protoplasts and stabletransformants.

Lehming et al. report that dramatic changes in binding affinity may beachieved by the modification of amino acids in the recognition helix ofthe lac repressor thus giving a tighter control of expression. Othersuch systems have been described and include the tetracycline induciblepromoter system developed by Gatz et al (1991, 1992) in which a modified35S CaMV promoter is repressed in plants expressing high levels of thetetracycline repressor protein but restored when tetracycline is added.

Steroid induction of protein activity can provide a chemically inducibleexpression system which does not suffer from chemical toxicity problems.Ligand binding domains of mammalian and insect steroid receptors such asglucocorticoid receptor (GR), oestrogen receptor can be used to regulatethe activity of proteins in mammalian cells (Picard et al. 1993). Aligand binding domain fused to a protein maintains the protein in aninactive state until the ligand is introduced. Lloyd et al. (1994)describes a fusion of a maize transcriptional regulator with GR. Simonet al. (1996) describe a fusion of GR with an Arabidopsis flowering timegene product responsible for induction of transcription and Aoyama etal. (1995) describe a fusion of Ga14 or VP16 with a planttransactivating protein, Athb-1, placed under steroid control by meansof the GR ligand-binding domain. It is known that the ability oftranscriptional activators to bind to DNA and to simultaneously activatetranscription is localised in defined domains of such transcriptionfactors. It has been demonstrated (Ptashne 1988) and Mitchell and Tijan(1989) that transcriptional activator factors are made up ofindependently finctioning modules. Ptashne and Gann (1980) and othershave shown that it is possible to combine a portion responsible fortranscription activation of one factor with a DNA binding portion ofanother factor and the resulting hybrid protein be active in yeastcells. A system incorporating these components may be used to relieverepression and thus induce expression of genes in a controlled manner.

The use of juvenile hormone or one of its agonists as a chemical ligandto control gene expression in plants by receptor mediatedtransactivation has also been described.

Preferably, the switch system used to inducibly express the restorergene is the AlcA/R switch system. We have demonstrated inducibleexpression of GUS in tomato anthers and pollen and have introducedsimilar constructs into wheat to demonstrate male and female tissue GUSexpression using the AlcA/R switch system. We have also demonstratedinducible GUS expression in maize tassels, silks, embryo and endospermnusing the safener inducible GST switch system (Jepson et al. 1994).Other switch systems may of course also be useful in the presentinvention.

The expression of cytotoxic or disrupter genes during planttransformation as a result of “leaky” expression from the male andfemale flower specific promoters at a very low level in tissues otherthan the target tissue may cause cell death and no recovery oftransformants.

The inducible promoters used in the present invention to driveexpression of the restorer genes may offer some protection against thispossibility for the following reasons.

In the case of the GST-27 promoter, constitutive expression has beenobserved in callus. In the case of the AlcA promoter which is inducedwith ethanol, induction of GUS expression has been observed atconcentrations of 7 ng/100 ml air. This concentration of ethanol can beadded to the tissue culture medium to ensure expression of the restorergene.

Examples of combinations of disrupter/restorer genes include barnase andbarstar, and TPP and TPS.

The use of translational enhancer sequences, in particular the TMV Ωsequence (Gallie et al.) is preferred in the present invention to givean increase in expression levels from the constitutive tissue specificand inducible promoters such that the expression of the restorer genee.g. barstar, is far in excess of that needed to inhibit the disruptergene e.g. barnase being produced. Gallie et al. showed that thetranslation of prokaryotic and eucaryotic mRNA's is greatly enhanced bya contiguous derivative of the 68 nucleotide, 5′ leader sequence oftobacco mosaic virus U1 strain called Ω. Several other viral leadersequences have also been shown to enhance expression, such as alfalfamosaic virus (A1MV) and broom mosaic virus (BMV). In tobacco mesophyllprotoplasts an enhancement of ˜20 fold was observed. Other enhancersequences e.g. tobacco etch virus may also be used in the presentinvention.

In addition, the use of intron sequences to enhance expression levels iswell documented. Among those studied are the maize adh 1 intron 1sequence which has been shown to increase levels of expression 12-20fold when inserted in 5′ translated sequences in chimeric constructsintroduced into maize protoplasts (Mascarenhas et al. 1990) and the Sh 1intron also from maize. The inclusion of this intron into constructs inwhich the CaMV35S promoter was driving CAT expression resulted inincreases of between 11 and 90 fold. (Vasil et al 1989).

Expression levels of the restorer and disrupter genes can also bebalanced or modulated in the following ways. A promoter giving highlevels of expression could be used to drive expression of the restorergene while a promoter giving lower levels of expression could be used todrive expression of the disrupter gene. This would ensure that thedisrupter gene product is swamped by restorer gene product therebyinactivating all cytotoxic or disrupter molecules allowing fullrestoration of fertility. A further way of modulating expression levelscould be carried out by using mutagenesis to change the sequence aroundthe AUG initiation codon in such a way that expression of the disruptergene is non-optimal (Kozak (1989)) and is therefore down-regulated.

The expression systems of the present invention can be introduced into aplant or plant cell via any of the available methods such asAgrobacterium transformation, electroporation, microinjection of plantcells and protoplasts, microprojectile bombardment, bacterialbombardment, particularly the “fibre” or “whisker” method, dependingupon the particular plant species being transformed. The transformnedcells may then in suitable cases be regenerated into whole plants inwhich the new nuclear material is stably incorporated into the genome.Both transformed monocot and dicot plants may be obtained in this way.Reference may be made to the literature for full details of the knownmethods.

Christou and Heie (1997) describe the transformation of rice usingbombardment methodology and progress on rice transformation mediated byAgrobacterium tumefaciens.

Other published methods for transforming wheat include Becker et al(1994) which describes the use of microprojectile bombardment ofscutellar tissue and Vasil et al (1993) which describes the rapidgeneration of transgenic wheat following direct bombardment of immatureembryos. FIG. 22 describes timelines for wheat transformation bybombardment.

The use of a selectable marker is required in the transformation processto select transformants carrying the sterility constructs. This could bean antibiotic selectable marker or a herbicide resistance gene. The useof a herbicide resistance gene or other marker is not essential (but maybe considered to be convenient) to the process of hybrid seedproduction.

The hemizygous plants used in the second method of the present inventioncan be treated with chemical to induce the expression of the restorergenes which allows self pollination to occur. The progeny of this selfpollination will be segregating and can be grown up, treated withchemical and self pollinated. The progeny from homozygous lines will notsegregate for sterility. A repeat of the process may then be performedto bulk up homozygous sterile seed.

The individual components of the expression cassette of the presentinvention may be provided on one or more individual vectors. These canbe used to transform or co-transform plant cells so as to allow theappropriate interaction between the elements to take place.

The present invention will now be described only by way of examples inwhich reference shall be made to the accompanying Figures:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic representation of expression cassettes used ina first method of hybrid seed production using a homozygous recessivemale sterile female parent.

FIGS. 2a and 2 b show the generation of F1 hybrid seed from a homozygousrecessive male sterile female parent and a female sterile male parentaccording to the first method of the present invention and thesegregation in the F2 generation.

FIG. 3 shows a schematic representation of expression cassettes used ina second hybrid seed production process.

FIG. 4 shows a schematic representation of an expression cassette usedin the production of a male sterile female fertile parent plant using amale flower specific promoter and a female tissue specific promoter.

FIG. 5 shows a schematic representation of an expression cassette usedin the production of a female sterile male parent plant using a maleflower specific promoter and a female flower specific promoter.

FIGS. 6a, 6 b and 6 c show the generation of F1 hybrid plants using amale sterile female parent plant and a female sterile male parent plant,both under the control of sporophytic promoters, according to the secondmethod of the present invention, the generation of the F1 hybrid seedand the segregation of the F2 progeny.

FIGS. 7a, 7 b and 7 c show the generation of F1 hybrid plants using amale sterile female parent plant and a female sterile male parent plant,male sterility being under the control of a gametophytic promoter andfemale sterility being under the control of a sporophytic promoter,according to the second method of the present invention, the generationof F1 hybrid seed and the segregation of the F2 progeny.

FIG. 8 shows the binary plant transformation vector pMOG1006.

FIG. 9 shows the binary plant transformation vector pMOG1006-FSE.

FIG. 10 shows the cloning vector pFSE4.

FIG. 11 shows GST expression in various maize tissues by northernanalysis.

FIG. 12 shows inducible expression of the GUS reporter gene in tobaccoleaf by the GST promoter.

FIG. 13 shows inducible expression of the GUS reporter gene in corn leafby the GST promoter.

FIG. 14 shows inducible expression of the GUS reporter gene in corntassels by the GST promoter.

FIG. 15 shows inducible expression of the GUS reporter gene in cornendosperm.

FIG. 16 shows the pPUG and RMS-3 cloning strategies.

FIG. 17 shows the pGSTTAK vector.

FIG. 18 shows RMS-3 vector.

FIG. 19 shows the map of pSRN.AGS, an inducible GUS expression vectorfor use in dicot species.

FIG. 20 shows the cloning strategy for pSRN.AGS, an inducible GUSexpression vector for use in dicot species.

FIG. 21 shows the map of pUIRN.AGS, an inducible GUS expression vectorfor use in monocot species.

FIG. 22 shows timelines for wheat transformation by bombardment.

FIG. 23 shows map of pSRN.

FIG. 24 shows map of pAGS.

FIG. 25 shows map of pMOG1006-SRN.AGS, a rice transformation vector.

FIG. 26 shows map of pGUN.

FIG. 27 shows map of pdvh405.

FIG. 28 shows Tap1AlcR-AlcAGluGUSIntnos.

FIG. 29 shows Stig1AlcR-AlcAGluGUSIntnos.

FIG. 30 shows corn transformation vector Zm/RMS14.

FIG. 31 shows pMOG1006-C5-GUS, a rice transformation vector.

FIG. 32 shows cloning vector pFSE.

FIG. 33 shows pMOG1006-MFS14-GUS, a rice transformation vector.

FIG. 34 shows Cassette A, MFS14-barase/barstar-nos.

FIG. 35 shows Cassette B, C5-barnase/barstar-nos.

FIG. 36 shows Cassette C, CaMV 35S-AlcR-nos.

FIG. 37 shows Cassette D, AlcA.Glul 1-barstar-nos.

FIG. 38 shows Cassette E, MFS14.Glul 1-barstar-nos.

FIG. 39 shows Cassette F, Stigl-barnase/barstar-nos.

FIG. 40 shows Cassette G, Stigl.Glul 1-barstar-nos.

FIG. 41 shows RMS30.

FIG. 42 shows RMS32.

FIG. 43 shows GUS expression in uninduced and ethanol induced wild typeand transgenic Alc-GUS tobacco callus.

FIG. 44 shows GUS expression in uninduced and ethanol vapour inducedwild type and transgenic Alc-GUS tobacco anthers.

FIG. 45 as above but induction by root drench with water and ethanol.

FIG. 46 shows GUS expression in uninduced and induced pistils from 9-10mm tobacco flower buds.

FIG. 47 shows GUS expression in uninduced and induced pistils from 17-22 mm tobacco flower buds.

FIG. 48 shows GUS expression in uninduced and induced pistils from 33-35mm tobacco flower buds.

FIG. 49 shows GUS expression in uninduced and induced oil seed rapeflowers.

FIG. 50 shows uninduced oilseed rape flower.

FIG. 51 shows GUS expression in oil seed rape pistils two days afterroot drench induction.

FIGS. 52 to 55 show GUS expression in stigma and style of ethanolinduced oil seed rape flowers.

FIG. 56 shows wild type and water induced Alc-GUS oil seed rape pistils.

FIG. 57 shows Alc-GUS oil seed rape pistils two days after 2% ethanolroot drench.

FIG. 58 shows water treated and 5% ethanol treated Alc-GUS pistils.

FIG. 59 shows oil seed rape flower after induction.

FIG. 60a shows GUS expression in tomato anthers, driven by the AlcApromoter and 60 b shows GUS expression in tomato pollen, driven by theAlcA promoter.

FIG. 61 shows the DNA sequence (SEQ ID NO: 29) encoding the ZmC5promoter sequence in maize.

The underlined A is the putative transcriptional start point and thebold and underlined ATG is the translational start point.

DETAILED DESCRIPTION OF THE INVENTION EXAMPLE 1

In this example, the male parent plant is male fertile and homozygousfemale sterile as a result of a natural or engineered mutation. Thefemale parent plant is homozygous recessive male sterile and femalefertile. The male sterility can be genetically manipulated or introducedby crossing or be due to a natural mutation. For example, it could bedue to a mutation in a gene such as MS45 which has been shown to act ina recessive manner, leading only to sterility when homozygous.

The parental lines also contain a DNA sequence encoding an induciblepromoter operably linked to a functional copy of the sterility gene thusrestoring fertility for the purpose of maintenance of the female andmale parental lines, respectively (see FIG. 1).

Crossing these two parent plants generates F1 hybrids which areheterozygous for the recessive male sterility and female sterilityalleles and are therefore fully fertile. If, however, the farmerharvests and grows the F1 seed, the F2 generation segregates forsterility leading to a loss of heterosis and yield as approximately 25%female plants are sterile (see FIGS. 2a, 2 b and 2 c).

Although female recessive mutations are known in corn, the genes havenot yet been cloned.

EXAMPLE 2

A second method of producing hybrid seed has been formulated based onsterility brought about entirely by genetic manipulation (see FIG. 3).

FEMALE PARENT

The female parent is a male sterile line i.e. an inactivating gene isexpressed by a male sporophytic promoter thereby preventing theproduction of viable fuictional pollen. In addition, a restorer gene isexpressed in the female tissues. Self fertility may be restored by meansof an inducible promoter linked to a restorer gene. Transformation withsuch an expression cassette (see FIG. 4) leads to hemizygous plants MSR^(F) R^(CS)----------.

R^(cs)=chemically restorable male and female fertility

MS=dominant male sterility

R^(M)=dominant male fertility restorer

FS=dominant female sterility

R^(F)=dominant female fertility restorer

To obtain homozygous plants for use in hybrid seed production, thehemizygous plants must be induced with an exogenous chemical inducer,such as ethanol in the case of the AlcA/R gene switch, and allowed toself-pollinate.

Gametes MS R^(F) R^(CS) ------------ MS R^(F) R^(CS) MS R^(F) R^(CS)------------ MS R^(F) R^(CS) MS R^(F) R^(CS) ----------- -----------—————— MS R^(F) R^(CS) ——————

Three quarters of the plants produce 100% sterile pollen as thesterility gene has a dominant effect, only the null line is malefertile. The homozygous plant can be distinguished from the heterozygousplants by growing up the seed resulting from self-pollination to repeatthe induction and self-pollination procedure. The progeny of these willsegregate for sterility and can be easily scored, depending on thegenotype, as described earlier. Herbicide resistance or other selectablemarker gene could also be used to score. In the case of the homozygousplant all progeny will be male sterile (and herbicide resistant,) in thecase of the heterozygous, the progeny will continue to segregate forsterility.

Thus, the homozygous male sterile, homozygous female fertile line can beselected in this way.

MALE PARENT

The male parent plant is fully male fertile but is female sterile i.e.homozygous female sterile, male fertile. In this case, the femalesterility is brought about by expressing, in the female floral organs,an inhibitory gene which is deleterious to female floral organdevelopment as defined earlier. Alternatively, the pollen tube may bedestroyed or the plant may otherwise be prevented from developing theseed. A restorer gene is also expressed in the male floral tissues.

The male parent plant is obtained in the same way as the female parentplant but using the construct shown in FIG. 5. In this case, thehemizygous plants havethe genotype:

FS R^(M) R^(CS)-----------

and the homozygous plants will have the genotype

FS FS R^(M) R^(M) R^(CS) R^(CS)

EXAMPLE 3

In this example, the expression cassette comprises a gametophytic (e.g.pollen specific) promoter (see FIG. 5). The male parent plant is asdescribed in Example 2 above. Examples of pollen specific promotersinclude Zm13 (Hanson et a.) and C5, deposited at The National Collectionof Industrial and Marine Bacteria as NCIMB 40915 on Jan. 26, 1998. TheC5 promoter sequence is shown in FIG. 61. Once again the homozygous malesterile line must be obtained and the hemizygous plants are chemicallytreated and self-pollinated.

Gametes MS R^(F) R^(CS) —————— MS R^(F) R^(CS) MS R^(F) R^(CS) MS R^(F)R^(CS) MS R^(F) R^(CS) ------------- —————— MS R^(F) R^(CS) ——————-------------- -------------

In this case, the homozygous line i.e. MS MS R^(F) R^(F) R^(CS) R^(CS)produces 100% sterile pollen which can be identified by DAPI staining.This can be distinguished from pollen from a heterozygous plant whichproduces 50% sterile and 50% fertile pollen which is also identifiableby DAPI staining.

Thus, the MS MS R^(F) R^(F) R^(CS) R^(CS) line can be selected.

It can therefore be seen that neither male or female homozygous parentcan self-pollinate.

The female parent plant in Examples 2 and 3 carries an expressioncassette comprising a female specific promoter driving the expression ofa restorer gene, and the male parent plant carries an expressioncassette comprising a male specific promoter driving the expression of arestorer gene. In the parent plants, these expression cassettes have noeffect on fertility due to the specificity of the promoters.

When these two parent lines are crossed, however, the result depends onwhether a gametophytic (e.g. pollen specific) or a sporophytic (e.g.tapetum specific) promoter has been used to produce male sterility inthe female parent. When a sporophytic promoter is used full restorationof fertility is achieved and the F1 seed is fully fertile i.e. producesapproximately 100% fertile pollen (see FIGS. 6a, 6 b). If, however, F1seed is retained and grown by the farmer, the sterility segregates asabove (see FIG. 6c). If a gametophytic promoter is used to obtain malesterility, full restoration of fertility is not achieved as pollen ishaploid and only about 50% of pollen will inherit the functional alleleproducing pollen fertility (see FIGS. 7a, 7 b). The F2 progeny segregateas described previously (FIG. 7c).

Maintenance of the homozygous sterile lines is achieved, when required,by the third component of the system. Each parental line comprises,therefore, an inducible promoter, optionally linked to an enhancersequence or one or more intron sequences, driving the expression of therestorer in all tissues on application of the inducing chemical, thusallowing pollen production in the female parent and normal ovule andtissue development in the, male parent. Self-pollination can then occurallowing bulking up of parental seed.

In all Examples, the application of chemicals in the field is necessaryonly to produce seed of the parental lines which needs to be doneinfrequently and on a relatively small acreage.

In the described Examples, it is possible to use a tapetum specific(FIGS. 6a, 6 b or 6 c) or a pollen specific promoter (FIGS. 7a, 7 b) todrive the expression of the inactivating gene. The use of a tapetumspecific promoter is preferred as fertility can be fully restored.However, as pollen is haploid, full restoration of fertility is notpossible where a pollen specific promoter is used. There may, however,be advantages to using a pollen specific promoter. For instance, pollenspecific promoters may have greater tissue specificity. Chemicaltreatment of the plants is only necessary to restore fertility to allowself-pollination.

EXAMPLE 4 PREPARATION OF GENERAL CLONING VECTORS

All molecular biology techniques were performed either as described byManiatis et al., Molecular Cloning: A Laboratory Manual second edition(1989) Cold Spring Harbour Laboratory Press: Vols I and II (D. N. Glovered 1985) or as recommended by the named manufacturers.

Preparation of pMOG1006-Fse

pMOG1006 (FIG. 8) is a binary vector which carries a hygromycinresistance gene as selectable marker and is used for Agrobacteriummediated transformation of rice. The modified vector was prepared bydigesting pMOG1006 with EcoRI and inserting an annealed pair ofcomplementary oligonucleotide having the sequences (SEQ ID NOS: 1-2):

Link1A AAT TGA TCG GCC GGC CCT AG

Link1B AAT TCT AGG GCC GGC CGA TC

which introduces a unique FseI site. Clones containing the correctoligonucleotide sequence were selected by hybridisation with link1Aoligonucleotide labelled with γ³²P and clones containing the sequence inthe desired orientation selected after characterisation by sequencing(FIG. 9).

pVB6

pVB⁶ is analogous to the binary vector described above in that itcontains a unique Fse1 site but carries the npt11 selectable marker andis used in Agrobacterium mediated tobacco transformation.

Preparation of DFse4 (FIG. 10)

This vector was constructed to allow the assembly of vectors containinga number of expression cassettes. This was achieved by using rare 8-baserecognition restriction enzyme sites in the multiple cloning siteregion. The pFSE (FIG. 32) DNA was digested with FseI and the existingmultiple cloning region removed. Complementary oligonucleotide havingthe sequences (SEQ ID NOS: 3-4):

DAA-1A:

5′ CCGTTTAAACATTTAAATGGCGCGCCAAGCTTGCGGCCGCCGGGAATTCGGCCGG-3′

DAA-1S: 5′ CCGAATTCCCGGCGGCCGCAAGCTTGGCGCGCCATTTAAATGTTTAAACGGCCGG-3′

were inserted. This sequence introduces a unique EcoRI, NotI, HindIII,AscI, SwaI and a PmeI site flanked by FseI sites. Expression cassetteswere assembled in pSK+ and linkers inserted to flank each cassette withunique sites as described below. Whole cassettes were then insertedpFSE4 as required. Multiple cassettes could then be removed as an FseIfragment to the pMOG1006-FseI and VB6 vectors described above. wereinserted. This sequence introduces a unique EcoRI, NotI, HindIII, AscI,SwaI and a PmeI site flanked by FseI sites. Expression cassettes wereassembled in pSK+ and linkers inserted to flank each cassette withunique sites as described below. Whole cassettes were then inserted intopFSE4 as required. Multiple cassettes could then be removed as an FseIfragment to the pMOG1006-Fse1 and VB6 vectors described above.

EXAMPLE 5 PCR CLONING OF THE STIGI PROMOTER FROM TOBACCO

A 1.6 kb fragment was PCR-amplified from 10 ng tobacco genomic DNA usingStratagene's PfuTurbo DNA polymerase and oligonucleotides (SEQ ID NOS:5-6) ST1-L2 (5′-ATTCGACCTCGCCCCCGAGCTGTATATG-3′) and ST1-R2(5′-GATGAGAATGAGAAGGTTGATAAAAGCC-3′). Thermocycling conditions were asfollows: 95° C. for 3 minutes, followed by 35 cycles of 95° C. for 1minute, 50° C. for 1 minute, 72° C. for 4 minutes, followed by a finalincubation at 72° C. for 5 minutes. A 1.6 kb amplification product wasgel-purified using QIAGEN's QIAquick Gel Extraction Kit and ligated intoInvitrogen's pCR-ZERO Blunt vector. The DNA sequence of the insert wasdetermined, and exhibited 100% identity with the published STIG1sequence (Goldman et al 1994). The insert was then transferred, on aSacI-NotI fragment, into pBluescript SK+ for further manipulation(pSK-STIG1).

EXAMPLE 6 PREPARATION OF PLANT TRANSFORMATION VECTORS TO TEST EXPRESSIONFROM CHEMICALLY INDUCIBLE PROMOTERS GST-GUS

The characterisation of the maize GST27 cDNA has been previouslyreported and experiments have shown that GST 27 is not constitutivelyexpressed in silks, leaf, embryo or endosperm. After safenerapplication, expression was detected in all of these tissues (see FIGS.11 to 15). Plant transformation constructs utilising the GST 27 promoterto drive the expression of GUS may be made as described in U.S. Pat. No.5,589,614 (FIG. 16). These are pGSTTAK (FIG. 17) for tobaccotransformation and RMS-3 (FIG. 18) for corn transformation. Thesevectors could be used to generate stable tobacco and maizetransforrnants. Formulated safener may be applied as either a leaf paint(tobacco) or as a root drench (corn) as described in the aforementionedpatent and expression of GUS observed. The results for induction of GUSexpression in tobacco leaves are shown in FIG. 12; clear induction ofexpression has occurred up to 100×. Similarly, for corn there has beenan induction of GUS expression in leaf after safener treatment.Induction of expression was also observed in tassels and endospermtissue and embryo.

RMS-3 was also used to transform wheat, and the induction of GUSexpression was studied. Modified vectors using hygromycin as aselectable marker could be introduced into rice.

GST-Barstar

The corn transformation vector Zm/RMS14 (described above) carries abarstar gene fused to the safener induced GST-27 promoter. WU25 wasshown by PCR to contain this gene fusion. Reversal of sterility wasdemonstrated by application by root drench of safener R-29148 to glasshouse grown plants. Treated plants showed increased tassel size relativeto untreated plants. There was no effect of the safener on the size orfertility of tassels on non-transgenic fertile plants. Correlated withincrease in tassel size, microspore development was observed in anthersamples taken from root drenched samples in the glass house. A similareffect was seen after a foliar application to field grown plants, butnot in untreated plants in any experiment. The resumption of microsporedevelopment appears to be linked to the barstar inhibition of barnasethus overcoming the ablation of tapetal cells. On plants exposed toextended treatment with safener, microsporogenesis had proceededresulting in anthers filled with immature post-mitotic pollen. Incontrast, the anthers from sterile plants were collapsed.

pSRNAGS

A binary plant transformation vector, pSRNAGS (FIG. 19) was constructedaccording to the strategy described in FIG. 20. This vector comprisesthe chimeric 35S-AlcA promoter driving the expression of GUS and the 35SCaMV promoter driving the expression of the AlcR gene.

pUIRN.AGS

A pUC based vector for use in transforming corn and wheat was preparedin which the ubiquitin promoter linked to the ubiquitin intron was usedto drive expression of the AlcR gene (FIG. 21). The time lines forobtaining transgenic wheat are shown in FIG. 22.

pMOG1006-SRNAGS (Rice)

The plasmid pSRN (FIG. 23) was digested with EcoRI and HindIII torelease a 2.6 kb fragment (AlcR-nos) which was cloned as an EcoRI-HindIfragment into pMOG1006. The 560 bp CaMV 35S promoter fragment was thencloned into the EcoRI site to produce 35S-AlcR-nos, clones in thedesired orientation were selected by sequence analysis(pSRN). Theplasmid pAGS (FIG. 24) was digested with Hindlll and a 2.5 kb fragment(AlcA-GUS-nos) was cloned into the HindIII site of pMOG 1006-SRN toproduce the final construct called pMOG1006-SRN-AGS (FIG. 25). Theorientation of the HindIII fragment was determined by restriction andsequence analysis.

In order to optimise levels of expression of AlcR in the tapetum,pistil, pollen and other reproductive tissues the following vectors wereprepared using tissue specific promoters to drive AlcR expression.

AlcA-Glu11-GUSint-nos

A SacI site at the end of the GUS gene in pGUN (FIG. 26) was changed toa PstI site using the QUIckChange Icit as usual and a subsequentNcoI-SacI digest containing the GUS gene (+intron) was used to replacethe barstar gene in cassette D (see later) to produce a vectorcontaining AlcA promoter-glucanase enhancer-GUS-nos. This was excised asa PmeI fragment and cloned into pFSE4.

Tap1-AlcR-nos-AlcA-Glu11-GUSint-nos

The AlcA-Glu11-GUSint-nos (Glu11 is glucanase 5′ untranslated region)was cloned as a PmeI fragment into PmeI cut pFSE4.

The tapetum specific Tap I promoter originally isolated from Antirrhinumand now cloned from pvdh405 (FIG. 27) was cloned as an EcoRI fragmentinto pSK-AlcR-nos (generated by cloning the EcoRI-HindIII fragment frompSRN into pSK+) and the resultant Tap1-AlcR-nos was cloned as a NotIfragment into pFSE4-AlcA-Glu11-GUSint-nos. The resultant FseI insert wasinserted into pVB6 to generate the final tobacco transformation vector(FIG. 28).

Stig1-AlcR-nos-AlcA-Glu11-GUSint-nos

This construct was made as above except that the pistil transmittingtract specific Stigl promoter cloned from tobacco (see below) was clonedas an EcoRI-NcoI fragment into pSK-AlcR-nos, and further manipulationsperformed as above. (FIG. 29).

EXAMPLE 7 CONSTRUCTION OF VECTORS TO ASSESS TISSUE SPECIFICITY OF MALEPROMOTERS pMS14-GUS

The 5.8 Kb promoter fragment from MFS14 (Wright et al. 1993) was fusedto the GUS reporter gene. Expression of GUS was detected only in anthersin the early stages of flower bud development but not in leaves.

pMS14-Barnase

The same promoter fragment was fused to barnase to generate the corntransformation vector ZM/RMS14 (FIG. 30). This fusion also contained anout of frame barstar gene with a functional bacterial promoter toprovide protection against barnase during the cloning steps. Transgenicmaize plants were obtained and several progressed for further analysis.One plant WU25 in particular was studied in some detail by analysingprogeny derived from crosses with pollen from a fully fertile plant. Allprogeny inheriting the transgene as assessed by PCR and leaf paint todetect the pat gene were completely sterile, whereas progeny lacking thetransgene were fully fertile. Sterile tassels were generally smallerthan those on fertile siblings and carried anthers that lacked pollenand did not excert from the tassel. In all other respects sterile plantswere indistinguishable from their fertile non-transgenic siblings.

pC5-GUS

A genomic clone of pectin methyl esterase from maize (named C5 as shownin FIG. 61) has been isolated and the promoter used in transcriptionalfusions with GUS to allow study of tissue specificity. The vector wasintroduced into tobacco by Agrobacterium transformation andtransformants selected on kanamycin. Pollen grains from dehisced antherswere harvested and stained for GUS activity. Two plants showedapproximately 50% blue staining pollen. No staining was seen innon-transgenic controls. Extracts were made from a range of tissuesincluding stages of developing anthers and analysed fluorometrically forGUS expression. Only very low levels of expression were seen in tissuesother than developing and dehisced anthers. Microspore stainingindicates that the timing of expression agrees well with Northern datawhich shows that both in transgenic tobacco and in maize, its nativeenvironment the ZmC5 promoter functions late in pollen development.

pMOG1006-C5-GUS (Rice)

C5-GUS(bin) was cut with EcoRI and BamHL to produce a 2.1 kb BamHI-EcoRIfragment (GUS-nos) which was cloned into EcoRI-BamHI cut pMOG1006 and a1.9 kb BamHI fragment (C5 promoter) which was subsequently cloned intothis pMOG1006-Gus-nos to produce the final vector, pMOG1006-C5-GUS,orientation of the promoter was confirmed by sequencing (FIG. 31).

pMOG1006-MFS14-GUS (Rice)

The 2.3 Kb MFS14 promoter was isolated from the RMS30 (FIG. 41) vectorusing BamHI and cloned into pFSE (FIG. 32). The GUS intron cassette wasthen cloned from pGUN as a PstI fragment into the pFSE-MFS14 vector. Thewhole MFS14-GUSint-nos fragment was then cloned as an Fsel fragment intopMOG1006-Fse (FIG. 33).

EXAMPLE 8 PREPARATION OF VECTORS TO GENERATE STERILITY AND RESTOREFERTILITY Preparation of Cassette A—MFS14-barnase-nos—a DominantSporothytic Male Sterility Cassette

The nos terminator from RMS14 was isolated as an EcoRI-HindIII fragmentand cloned into pSK+ cut with the same two enzymes. The resultingplasmid was digested with HindIII and an annealed pair of complementaryoligonucleotide having the sequences (SEQ ID NOS: 7-8):

Link2A AGC TTC TGG AAT TCG TCT

Link 2B AGC TAG ACG AAT TCC AGA

i.e. coding for dHindIII-EcoRI-HindIII ligated with the cut vector.Putative transformed colonies were streaked into nylon membranes andprobed in the usual way with the Link2A oligonucleotide labelled withγ³²P. A number of positive colonies were analysed by sequencing and oneclone having the orientation in which the HindIII site was internalrelative to the two EcoRI sites selected for further manipulation. Intothis vector, cut with HindIII and treated with shrimp alkalinephosphatase to prevent religation of the vector was ligated a HindIIIfragment isolated from RMS14 carrying the MFS14 promoter and the barnaseand barstar coding sequences. Tranformants containing the HindIIIfragment in the desired orientation were identified by sequence analysis(FIG. 34). The entire fragment carrying MFS14-barnase/barstar-nos wasexcised on an EcoRI fragment and inserted into pVB6 and pMOG1006-Fse viapFse4 for introduction into rice and tobacco.

Preparation of Cassette B—C5-barnase—a Dominant Gametophytic MaleSterility Cassette

The unique SalI site of pBluescript SK+ (Stratagene) was replaced with aNotI recognition site by insertion of the an oligonucleotide (SEQ ID NO:9) linker MKLINK4 (5′-TCGATTCG GCGGCCGCCGAA-3′) into the digested SalIsite. A 0.9 kb, BamHI-HindIII fragment carrying the coding region ofbamase followed by a bacterial-promoter-driven barstar coding region,was inserted into the corresponding fragment of the modifiedpBluescript. The nos terminator on a HindIII-NotI fragment was insertedinto the corresponding fragment of the resulting vector. An unwantedBamHI site was then removed using Stratagene's QuickChange system,following the manufacturer's instructions and using oligonucleotides(SEQ ID NOS: 10-11) DAM-3A (5′GGTCGACTCTAGAGGAAC CCCGGGTACCAAGC-3′) andDAM-3S (5′-GCTTGGTACCCGGGGTTCCTCTA GAGTCGACC-3′). The resulting plasmid(named pSK-BBN) was digested to completion with BamHI, dephosphorylatedwith shrimp alkaline phophatase (37° C., 1 hour). A 1.9 kb BamHIfragment of the C55′ flanking region was ligated into this, followed bydigestion with BamHI and PstI to check for presence and orientation ofthe insert, respectively. The resulting plasmid was named pSK-C5-BBN(FIG. 35). The entire cassette was then removed as an EcoRI-NotIfragment to a binary plant transformation vector pVB6. The construct wasthen introduced into Agrobacterium Tumefaciens by the freeze-thawmethod. Standard techniques were used to introduce the DNA into tobacco.

Prerparation of Cassette C—35S-AlcR-nos

An EcoRI-HindIII fragment was isolated from the vector known as pUC3,this fragment contains the AlcR coding sequence and nos terminator. Thisfragment was cloned into EcoRI-HindIII cut pSK+. An annealed pair ofcomplementary oligonucleotides having the sequences (SEQ ID NOS: 12-13):

Link5A AGC TAT TAG CGG CCG CTA TGT TTA AAC GCG T

Link5B AGC TAC GCG TTT AAA CAT AGC GGC CGC TAA T

carrying restriction sites for ΔHindIII-NotI-PmeI-ΔHindIII was insertedinto the HindIII site, thus adding a Pme1 and Not1 site and deleting theHindIII site at the 3′ end of the cassette. The EcoRI fragment from pUC3carrying the 35S CaMV promoter was cloned into the EcoRI site andoriented by restriction and sequence analysis (FIG. 36). The entirecassette can be excised as a NotI fragment for further manipulation, andcontains the PmeI site into which the AlcA-Glu11-barstar-nos cassettecan be inserted.

Preparation of Cassette D AlcA-Glu11-barstar-nos

The vector pMJB1 was digested with XhoI and NcoI to remove the TMV omegaenhancer. Two oligonucleotides encoding the glucanase 11 5′UTR andflanked by XhoI and NcoI sites, having the sequences (SEQ ID NOS:14-15):

Glu1 TCG AGA CAA TTT CAG CTC AAG TGT TTC TTA CTC TCT CAT TTC CAT TTT AGC

Glu11 CAT GGC TAA AAT GGT TTT GAG AGA GTA AGA AAC ACT TGA GCT GAA ATTGTC

were annealed as usual and ligated into the cut vector. The sequencedvector was digested with HindIII and XhoI to remove the 35S CaMVpromoter and the AlcA promoter ligated in on a HindIII-XhoI fragment. Afragment encoding barstar having NcoI and BamHI ends was obtained by PCRfrom RMS9. This was then inserted into the above vector cut withNcoI-BamHI to give a complete cassette carrying AlcA-Glu11-barstar-nos.In order to facilitate manipulation of the cassette a PmeI site wasadded at each end by the use of oligonucleotides encodingΔHindIII-PmeI-HindIII and ΔEcoRI-PmeI-ΔEcoRI (FIG. 37). This allows theinsertion of this cassette into the 35S-AlcR-nos cassette describedabove enabling both components of the AlcA/R switch to be moved intopFSE4 as a NotI fragment.

Preparation of Cassette E MFS14-Glu11-barstar-nos—a Male FertilityRestorer Cassette

A 320 bp BamHI-SacI fragment carrying the 3′ end of the MFS14 promoterwas cloned into BamHI-SacI cut pSK+. The HindIII site was removed usingthe QuickChange kit from Qiagen following standard procedures. Theoligonucleotides used to remove the site had the sequences (SEQ ID NOS:16-17):

MKM1A CGG TAT CGA TAA GCT AGA TAT CGA ATT CCT G

MKM1S CAG GAA TTC GAT ATC TAG CTT ATC GAT ACC G

The deletion of the site was confirmed by restriction analysis and bysequencing. A new HindIII site was then introduced near the SacI site byinsertion of annealed oligonucleotides (SEQ ID NOS: 18-19) encodingΔSacI-HindIII-SacI sites.

SHSLINK1 CAT AAA GCT TAT ACA GCT

SHSLINK2 GTA TAA GCT TTA TGA GCT

The presence of the new site was confirmed by restriction analysis andthe correct orientation of the linker defined by sequencing. The desiredorientation gave a HindIII site to the outside of the SacI site relativeto the BamHI site. The BamHI site was then removed and a new XhoI siteintroduced in the same way using an oligonucleotide encodingdBamHI-XhoI-ABamHI. This had the sequence (SEQ ID NO: 20):

Link6 GAT CGT ATC TCG AGA TAC

The absence of the BamHI site and the introduction of the XhoI site wasconfirmed in the usual way.

RMS14 was digested with HindIII and SacI and the 5.5 Kb fragmentencoding the remainder of the MFS14 promoter was isolated. This fragmentwas then inserted into HindIII-SacI cut vector above and the integrityof the promoter confirmed by sequencing. The whole of the MFS14 promoterwas then excised as a HindIII-XhoI cassette and inserted in Cassette Dreplacing the AlcA promoter at the stage before addition of linkers tothe ends. For this cassette linkers introducing Swa1 sites at each endwere used (FIG. 38).

Preparation of Cassette F Stig1-barnase-nos—a Female SporophyticSterility Cassette

A BamHI site was introduced close to the translation start site of theSTIG1 promoter in the vector pSK-STIG1. This was achieved using theStratagene QuickChange Kit with oligonucleotides (SEQ ID NOS: 21-22):

ST1-BA (5′-GATAAAAGCCATAATTGGATCCTGGTGGTTTCTGC-3′) and

ST1-BS (5′-GCAGAAACCACCAGGATCCAATTATGGCTTTTATC-3′).

The 1.6 kb SUG1 promoter was then released on a BamHI fragment andcloned into BamHI-digested pSK-BBN. Presence and correct orientation ofthe promoter were determined by PCR amplification between vector andpromoter sequences. The STIG1-BBN cassette was transferred, on aNotI-EcoRI fragment to the vector pFSE4, the resulting plasmid beingnamed pFSE4-STIG1-BBN (FIG. 39). The entire cassette was thentransferred as an FseI fragment to VB6.

Preparation of Cassette G Stig1-Glu11-barstar-nos—a Female FertilityRestorer Cassette

The construct pAlcA-GluII-barstar-nos-pp was modified to replace aHindIII restriction site with an EcoRI site. This was achieved using theStratagene QuickChange Kit and oligonucleotides (SEQ ID NOS: 23-24):

DAM-6A (5′-CGGAACTATCCCGAATTCTGCACCGTTTAAACGC-3′) and

DAM-6S (5′-GCGTTTAAACGGTGCAGAATTCGGGATAGTTCCG-3′).

A XhoI site was introduced close to the translation start site of theSTIG1 promoter in the vector pSK-STIG1. This was achieved usingStratagene's QuickChange Kit with the oligonucleotides (SEQ ID NOS:25-26):

ST1-XA (5′-GATAAAAGCCATAATTGGCTCGAGGTGGTTTCTGCTGAG-3′) ST1-XS(5′-CTCAGCAGAAACCACCTCGAGCCAATTATGGCTTTTATC-3

The 1.6kb STIG 1 promoter was then released on a XhoI-EcoRI fragment andcloned into the larger fragment produced by digestionpAlcA-gluII-barstar-nos-pp with XhoI and EcoRI, replacing the AlcApromoter with the STIGI promoter (FIG. 40). The whole cassette wasexcised on a PmeI fragment and cloned into pVB6 and pMOG1006-Fse.

Preparation of a Switchable TPS Vector—AlcA-TPS-nos Tap1-AlcR-nos

The Tap1-AlcR-nos cassette has been described. The GUS gene in pAGS wasreplaced with TPS and the new cassette was cloned as a HindIII cassetteinto pFSE4. The Tap1-AlcR-nos described earlier was as a NotI fragmentinto this pFSE4-AlcA-TPS-nos vector. The whole Fsel fragment was excisedand cloned into a pVB6. This vector can now be used to retransformtobacco made sterile by transformation with a construct carrying aTap1-TPP-nos expression cassette.

EXAMPLE 9 PREPARATION OF PLANT TRANSFORMATION VECTORS

Any combination of the above cassettes may be made in pFSE4 andsubsequent transfer of the resulting Fsel fragment into eitherpMOG1006-Fse or pVB6 allows transformation into tobacco or rice.

The following combinations of cassettes have been made as planttransformation vectors,

A+C+D=sporophytic male sterile plant whose fertility is restorable bychemical induction of the restorer gene

E=sporophytic male fertility restorer plant, which when cross pollinatedonto the above plant restores fertility in the progeny

F+C+D=sporophytic female sterile plant whose fertility is restorable bychemical induction of the restorer gene

G=sporophytic female fertility restorer plant which when crosspollinated by the above plant produces female fertile progeny.

B+D=gametophytic male sterile plant whose fertility can be restored bychemical induction of the restorer gene

The generation of the parents of F1 hybrid plants as described herein isachieved in the following way:

Male Parent i.e. female sterile Cassette F Stig1-barnase femalesterility Cassette E MFS14-Glu11-barstar male restorer Cassette C35S-A1cR-nos switch component Cassette D AlcA-Glu11-Barstar “ ” Femaleparent i.e. male sterile Cassette A MFS14-barnase male sterilityCassette G Stig1-Glu11-barstar female restorer Cassette C 35S-AlcR-nosswitch component Cassette D AlcA-Glu11-barstar “ ”

EXAMPLE 10 CONSTRUCTION OF VECTORS TO TEST NEW PIG GENE RMS30 and RMS32(Tobacco)

The tubulin gene was cloned as a Hinf1 fragment from ptubulin and clonedinto pFSE-MFS14 generated above. The resultant MFS14-tubulin nos wascloned into pVB6 as an FSE fragment to produce RMS30 (FIG. 41).

The MFS14+lac operator promoter was excised from RMS32 as a BamHIIfragment and cloned into pFSE. Following insertion of the tubulin gene,the MFS14-lac-op-tubulin-nos FseI fragment was cloned into pVB6 toproduce RMS32 (FIG. 42).

RMS30 and RMS32 (Rice)

The FseI fragments containing MFS14 promoter (+/− lac op)-tubulin-noswere cloned into pMOG1006-Fse to generate the plant transformationvectors.

Optimisation of Trehalose-phosphate Phosphatase(TPP) andTrehalose-6-phosphate Hydrolase (TreC) for use in Generating Sterilityin Zea Mays

Versions of TPP and TreC coding regions optimised for expression in Zeamays (which has a preference for G or C in redundant positions of eachcodon) are synthesised by Operon Technologies Inc. Nucleotide sequencesare derived from their amino acid sequences using codons present in vivoabove 1.0% and at frequencies representative of the naturally occurringratios (according to the Genbank Codon Usage Database, Release 108).Also included are useful restriction enzyme sites close to thetranslation start sites including BamHI and NcoI and, and a PstI site atthe 3′ ends to facilitate cloning.

Constructs to Test the use of Codon Optimised TPP and TreC Zea Mays

The BamHI site at the 5′ end of the MFS14 promoter in the vectorpFSE-MFS14 is removed using Stratagene's QuickChange system with theoligonucleotides (SEQ ID NOS: 27-28):

DAM-7A 5′-CGATGCTTTCGGAACCGGTACCGAATTCG-3′

DAM-7S: 5′-CGAATTCGGTACCGGTTCCGAAAGCATCG-3′

The synthetic TPP and TreC genes are then excised on BamHI—PstIfragments and cloned between the MFS14 promoter and nos terminator inthe modified pFSE-MFS14 vector. The adh1 intron is inserted between thepromoter and coding sequences to boost expression levels. The completecassette is then transferred to a pUC based cloning vector containingIGPD bacterial selection marker and a herbicide resistance gene cassettefor selection of transgenic plants.

EXAMPLE 11 PRODUCTION OF TRANSGENIC PLANTS

pSRN.AGS has been introduced into tobacco, oilseed rape and tomato(these plants are referred to as Alc-GUS plants) pMOG1006-Fse-SRNAGS andpMOG1006-C5-GUS have been introduced into rice via Agrobacteriummediated transformation.

Plant transformation vectors containing each of the cassettecombinations given above have been introduced into tobacco and/or riceby Agrobacterium mediated transformation. Explants were analysed by PCRand those containing intact inserts were clonally propagated andtransferred to the glass house and grown to flowering. RMS30 and 32 havealso been introduced into tobacco.

pUIRN.AGS has been introduced into wheat and corn via projectilebombardment methods, and transgenic plants recovered, by means ofco-bombardment with a plasmid carrying a selectable marker. Othervectors to test various components are transformed into corn bymicroprojectile bombardment.

EXAMPLE 12 ANALYSIS OF TRANSGENIC PLANTS Studies on GUS Expression Fromthe AlcA Promoter In Tissue Culture

In order to assess whether plants, following transformation with aconstruct containing a promoter attached to a cytotoxic gene, such asbarnase, would be recoverable, particularly if the promoter has someexpression in the callus phase of the transformation process, expressionin tobacco callus was studied to determine whether or not expression wasconstitutive as in the case of the GST27 promoter, or whether it couldbe induced by application of ethanol.

Four-week old Alc-GUS (35S-AlcR-nos/AlcA-GUS-nos) tobacco plants grownunder standard tissue culture conditions were used to test theexpression of the AlcA promoter in callus both with and without ethanol.Leaf discs were produced and placed onto MS medium supplemented with 3%(w/v) sucrose and 0.8% (w/v) Bacto-agar, 1 μg/ml 6-BAP and 100 ng/ml NAAhormones. Some of the discs were placed onto this medium containing 0.1%(v/v) ethanol. After 3 weeks when callus production had progressed,samples of callus were used for fluorometric GUS assays, the result ofwhich is shown in FIG. 43.

The GUS levels show that the AlcA promoter is leaky in callus and levelscan be increased with the addition of ethanol to the plant tissueculture medium. Therefore, transgenics may be recovered using the AlcApromoter driving a restorer gene in the same construct as a promoterdriving a cytotoxic gene.

In the Glasshouse

The AlcA/R gene switch has been demonstrated to give good levels ofreporter or trait gene induction in the leaves of tobacco upon additionof the chemical inducer ethanol, either by application as spray, vapouror root drench. (Caddick et al., Salter et al.) Transgenic AlcA-GUStobacco, oilseed rape and tomato plants were used to examine geneinduction in floral tissues.

Tobacco

1) A plastic bag was sealed around a tobacco AlcA-GUS tobacco flowerstem and a small pot containing 50 ml of 5% ethanol was placed insidethe bag. After 3 days anthers from different flower bud stages wereharvested and assayed for GUS expression. Results are shown in FIG. 44.This shows the GUS values from four independent flower buds from wildtype, uninduced AlcA-GUS and induced AlcA-GUS plants. Flower buds weremeasured in mm and each bar represents five anthers. The graph showsthat compared to AlcA-GUS plants which did not receive the ethanolvapour treatment, anthers from the induced plant contained higher levelsof GUS in them.

2) Flower stems from wild type and Alc-GUS tobacco plants were cut andplaced into beakers containing 300 ml of either water, 1%, 2% or 5%ethanol and left in the glasshouse for two days before harvestinganthers from the flower buds for fluorometric GUS assays. FIG. 45 is abar graph representing the GUS data from this experiment showing anthersfrom wild type, water treated Alc-GUS flower stem and 1%, 2% or 5%ethanol treated Alc-GUS flower stems. This experiment also demonstratesthat expression of the reporter gene has been induced by ethanol in theanthers at levels above those seen in the water treated flowers. Levelsof GUS expression from the induced AlcA-GUS anthers were above thosefrom a MFS14-GUS plant.

3) Mature AlcA-GUS tobacco plants were root drenched in the glasshousewith 250 ml of either water, or 5% ethanol. A wild type and a 35S-GUSplant were also treated with ethanol. Two days later pistils fromvarious size flower buds were dissected and stained for GUS activity.FIG. 46 shows pistils from 9-10 mm buds from the water and the 5%ethanol treated Alc-GUS plants. The photograph clearly demonstrates thatthe ethanol treatment has lead to induction of GUS in the pistils. FIGS.47 and 48 show the stigma/style region of pistils from 17-22 mm and33-35 mm buds respectively, again from water treated and ethanol treatedAlc-GUS plants. GUS staining is present throughout this area compared tothe water treated plant which was similar to wild type.

4) Alc-CAT plants (35S-AlcR-nos/AlcA-CAT-nos) were also tested andshowed an increase in reporter gene levels in floral tissues afterinduction with ethanol.

Oilseed Rape

1) Oilseed rape (OSR) AlcA-GUS plants were root drenched with 250m1 ofeither water, 1% or 2% ethanol both on day 0 and day 1. Flower samplesfrom these plants were then taken two days after the first induction.The samples taken for fluorometric GUS analysis were anthers from matureflowers (mature indicating that they were fully opened), stigma/stylesfrom mature flowers, anthers from immature flowers (flower buds with thepetals unopened), stigma/styles from immature flowers and finally therest of the flower pistils which includes the ovaries.

FIG. 49 shows the GUS data graphically and from left to right shows theresults from water induced Alc-GUS, 1% ethanol induced Alc-GUS and 2%ethanol induced Alc-GUS oilseed rape plants. The first two bars in eachsection represent uninduced anther and uninduced stigma/style GUS levelsrespectively. Each bar represents three replicates with each replicatecontaining anthers from three different flowers or stigma/styles fromeight different flowers or ovaries from six different flowers.

The data clearly shows an increase in GUS levels in floral tissues whencomparing the water treated oilseed rape to the ethanol-induced plants.All tissues examined in the 2% ethanol-treated plants show an increasein GUS from both uninduced levels and from the water-treated controlplant.

2) Oilseed rape Aic-GUS flowers were subjected to GUS staining followinginduction with ethanol. FIG. 50 is a photograph of an Alc-GUS flowerbefore induction and FIG. 51 is a photograph of a flower from the sameplant, two days after root drenching with 250 ml of 5% ethanol. Thisshows that the treatment has lead to reporter gene induction in thestigma/style region as well as the filaments.

FIG. 52 shows immature flower buds with the sepals and flowers removed.The ethanol treated plant on the right shows GUS expression in thestigma/style region compared to the water treated control on the left.FIGS. 53-55 are further examples of this. FIG. 56 shows pistil sectionsfrom wild type and water induced oilseed rape Alc-GUS plants. FIG. 57shows pistil sections from a plant root drenched with 2% ethanol twodays before the flower samples were taken. This shows induction of GUSthroughout the pistil region. FIG. 58 shows pistils from a water-treatedplant and a 5% ethanol treated plant. Again it is shown that theapplication of the chemical ethanol has led to the induction of GUS inthe female tissues.

FIG. 59 shows a flower from an experiment where a flower stem was cutand placed into a beaker of 5% ethanol and left for two days beforestaining the whole inflorescence for GUS activity. Blue staining isapparent in the filaments, sepals, petals and the stigma/style regionsof the flower.

Tomato

Alc-GUS tomoato flowers were induced using ethanol vapour as describedabove for tobacco flowers. Two days after induction, anthers and pollenwere stained to detect GUS expression. As can be seen in FIGS. 60a and60 b, blue staining was observed in both tissues.

Rice

Early experiments to test the inducibility of the Alc switch in riceinclude a hydroponics test involving leaf discs exposed to ethanolvapour for two days before assaying for GUS activity. Once in theglasshouse, whole plants are root-drenched with 1-5% ethanol before andduring pannicle formation/flowering phase to investigate induction ofGUS expression in the flower tissues.

GUS Assays

Flower material was ground in 2-300 μl of extraction buffer (100 mMsodium phosphate buffer pH 7, 10 mM EDTA, 0.1% Triton X-100, 1%Sarcosyl, 10 mM b-mercaptoethanol). Samples were spun in amicrocentrifuge for 15 min and 5 μl of the supernatant was used forBradford protein assays with BSA as standard. 20 μl was diluted 1 in 5with extraction buffer and 400 μl of assay buffer (same for extractionbuffer except contains 1 mM 4-MUG and 20% methanol). 100 μl was taken(T₀ sample) and added to 900 μl of stop buffer (0.2M sodium carbonate)and the rest was incubated at 37° C. for two hours before taking afurther 100 μl (T₂ samples) to be added to 900 μl stop buffer.

Florescence of samples was measured in a spectrophotometer and GUS wasrepresented as nM 4M U per mg protein per hour.

Wheat

Scutellum tissue is bombarded with pUIRN.AGS and the tissue exposed toethanol vapour. Staining for GUS expression is performed after 2-3 days,when numerous blue spots are seen indicating that the AlcA promoter isinduced leading to GUS expression. Transgenic Alc-GUS plants obtainedare grown to maturity and seed harvested, inducible GUS expression isdetermined on the progeny plants in the same way as described above forrice.

GUS Histochemistry

For the GUS staining, the protocol of Blume and Grierson (1997) wasused.

Investigation of Sterility

1)Male Sterile Plants

a) Sporophytic male sterile plants generated by transformation withcassette A are identified by the lack of pollen or by the presence ofdead pollen. Seed is produced by back crossing with wild type tobacco aspollen donor.

b) Gametophytic sterile plants generated by transformation with cassetteB are identified using vital stain on the pollen, 50% of the pollen issterile, 50% fertile.

2)Female Sterile Plants

Sporophytic female sterile plants generated by transformation withcassette F are identified by their inability to be cross pollinated withwild type pollen. Restorer plants in both cases generated bytransformation with cassettes E and G are self pollinated, progeny grownand homozygous lines selected in the usual way by selection on kanamycinof T2 seed.

Inducible Restoration of Fertility

The progeny of the cross between sporophytically sterile plants and wildtype plants are expected to segregate 1:1 for sterility with thepresence of the transgene and are selected at an early growth stage byPCR analysis. Induction experiments are performed to investigaterestoration of fertility, as these sterile plants can be treated withethanol via a root drench or spray or vapour application to induceexpression of barstar in the relevant tissues. It is expected thatinduction at the appropriate time allows self pollination to occur andseed production follows which can be easily scored. Homozygous plantsare selected from the resultant progeny by the usual means and used totest the constitutive restoration by crossing with homozygous restorerplants.

Back crossing the C5-barnase.AlcA-barstar plants with wild type plantsor allowing self pollination to proceed, however, results in therecovery of a population, 50% of which are fully fertile and 50%producing pollen of which only 50% is fertile. These plants can howeverbe induced with an ethanol spray, root drench or vapour treatment toexpress barstar in the developing pollen to allow self pollination. Thehomozygous plants in this case are 100% sterile, whereas the hemizygousplants are 50% sterile. The differences in results obtained by stainingallows conclusions to be drawn about the efficiency of induction andself pollination.

Constitutive Restoration of Fertility by Cross Pollination

The crossing of the male sterile plants with the male restorer plant isachieved by transferring pollen from the restorer plant to the pistil ofthe male sterile plant (after removal of any anthers that are presenteven though these will contain only dead pollen.) The pollinated pistilis then bagged to prevent contamination by wild type or other pollen inthe environment. Seed produced is harvested. Progeny are grown and theproduction of pollen is observed and measured. Restoration of fertilityin this way leads to normal pollen production.

The crossing of the female sterile plants is achieved by transferringpollen from these plants to the pistils of the female fertility restorerplants, again after removal of the anthers from these plants. The floweris bagged as above. Seed produced is harvested. The progeny are grownand the flowers bagged. The ability of these plants to self pollinate isobserved. Restoration of fertility in this way allows self pollinationto occur. In the same way the plants generated by transformation withcassettes E and F ie female sterile and carrying the male restorer geneand cassettes A and G ie male sterile and carrying the female restorergene are analysed.

Other modifications to the present invention will be apparent to thoseskilled in the art without departing from the scope of the invention.

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29 1 20 DNA Artificial Sequence oligonucletoide 1 aattgatcgg ccggccctag20 2 20 DNA Artificial Sequence oligonucletoide 2 aattctaggg ccggccgatc20 3 55 DNA Artificial Sequence oligonucletoide 3 ccgtttaaac atttaaatggcgcgccaagc ttgcggccgc cgggaattcg gccgg 55 4 55 DNA Artificial Sequenceoligonucletoide 4 ccgaattccc ggcggccgca agcttggcgc gccatttaaa tgtttaaacggccgg 55 5 28 DNA Artificial Sequence oligonucletoide 5 attcgacctcgcccccgagc tgtatatg 28 6 28 DNA Artificial Sequence oligonucletoide 6gatgagaatg agaaggttga taaaagcc 28 7 18 DNA Artificial Sequenceoligonucletoide 7 agcttctgga attcgtct 18 8 18 DNA Artificial Sequenceoligonucletoide 8 agctagacga attccaga 18 9 20 DNA Artificial Sequenceoligonucletoide 9 tcgattcggc ggccgccgaa 20 10 32 DNA Artificial Sequenceoligonucletoide 10 ggtcgactct agaggaaccc cgggtaccaa gc 32 11 32 DNAArtificial Sequence oligonucletoide 11 gcttggtacc cggggttcct ctagagtcgacc 32 12 31 DNA Artificial Sequence oligonucletoide 12 agctattagcggccgctatg tttaaacgcg t 31 13 31 DNA Artificial Sequence oligonucletoide13 agctacgcgt ttaaacatag cggccgctaa t 31 14 51 DNA Artificial Sequenceoligonucletoide 14 tcgagacaat ttcagctcaa gtgtttctta ctctctcatttccattttag c 51 15 51 DNA Artificial Sequence oligonucletoide 15catggctaaa atggttttga gagagtaaga aacacttgag ctgaaattgt c 51 16 31 DNAArtificial Sequence oligonucletoide 16 cggtatcgat aagctagata tcgaattcctg 31 17 31 DNA Artificial Sequence oligonucletoide 17 caggaattcgatatctagct tatcgatacc g 31 18 18 DNA Artificial Sequence oligonucletoide18 cataaagctt atacagct 18 19 18 DNA Artificial Sequence oligonucletoide19 gtataagctt tatgagct 18 20 18 DNA Artificial Sequence oligonucletoide20 gatcgtatct cgagatac 18 21 35 DNA Artificial Sequence oligonucletoide21 gataaaagcc ataattggat cctggtggtt tctgc 35 22 35 DNA ArtificialSequence oligonucletoide 22 gcagaaacca ccaggatcca attatggctt ttatc 35 2334 DNA Artificial Sequence oligonucletoide 23 cggaactatc ccgaattctgcaccgtttaa acgc 34 24 34 DNA Artificial Sequence oligonucletoide 24gcgtttaaac ggtgcagaat tcgggatagt tccg 34 25 39 DNA Artificial Sequenceoligonucletoide 25 gataaaagcc ataattggct cgaggtggtt tctgctgag 39 26 39DNA Artificial Sequence oligonucletoide 26 ctcagcagaa accacctcgagccaattatg gcttttatc 39 27 29 DNA Artificial Sequence oligonucletoide 27cgatgctttc ggaaccggta ccgaattcg 29 28 29 DNA Artificial Sequenceoligonucletoide 28 cgaattcggt accggttccg aaagcatcg 29 29 1891 DNAArtificial Sequence ZmC5 promoter sequence in maize 29 ggatcctgaaacatatcagt tgtgtttgtt tttgtaaatc ttttatacta ctaggggaga 60 aaattagcttagttcaatcg catctcatat gtctaattac caggggagaa aattagctta 120 gttcattttgttgctgccat atggggtgaa aaaataatga gacatctaaa tcagtaaatt 180 ggaaatatagcatcttaaac ctgcaggtag tttcttaaac ctgattctag ctacaactta 240 gtacaactactggtagtttt ttaaacctga ttctagctac atgttttata ttgtggcaca 300 agaacttttaagaacatatg ctgatgccca ctgtatttag ttactacttc aagaccaact 360 gtattttagttacaaatgtg ttttcaagat tgtagaaatt tgtagctgaa attatccaca 420 ccatatttgtgaactgacat catttctaag aatattactg attagaatct ttcactttta 480 taatgctttgcaggagtggc ccctctggag ttgaatatgc agttataacc aaattttacc 540 ccttttatcctagaagagtt gccaagacac ggtataagac catgataata gactaagaga 600 ggatttggctctaattacta tatgttttat ttatgcagtc ccatgagaac tttgagtatt 660 tgcagattgctttattaatt tattaaagtt aaagattgta tgtgttgagt atgtatccac 720 tcttgttggaagtgtcttgc aattccaatc caaggatgta taaaatactg catgggctaa 780 gtatgtgttttttcatgtat ttggagtata tatacttttt gttgcttgag aacatgtatg 840 tacactagaagcttgtcaat tgtgtgaact tgagttgatc cctgtctaac ctgagtatat 900 atatatatatattttgttgc ttgagaacaa gtatgtacaa tagaggcttg acaattgtgt 960 gaacttgagttgaacatgaa ttttgataat cacaactcac catccctttc aatatgctta 1020 gaatatagctttttataatt tttcacccta caatacaaaa ttgttctatg aaggccatgg 1080 tacatcatcatatcctgtat tatcaaccta ggatttgtct atttcgatta ataatggcat 1140 tgagtcaaattttggttgtt tcaaatgata gacttcgata tttgttatga tttatgagtt 1200 gattcttgatagcattacta aaaaatgacc tatgtatata caagtgtctt ccgttgcaac 1260 gcacgggcatatacctagtc aatcactaag accctaattt tgaagttggg acttagacgt 1320 gttccacgtttgtaaaggcg agtatatagg tgtatgtata taagagccgg tgtatacaac 1380 aattttttataagaaaactt gaacaagtag ccaggtgttg aaatcttcat atatgtgccg 1440 acgccattcaacatcatatt tggcttctgg cgaggatcgt agtatcaagc aacataaaag 1500 caatgacaaacagcgaagca caaagatctc ccaggctcgt cataaactaa tcacaatgtt 1560 gtttgtcctccacaattagc acaacccatt ttagaaaaag atgccacgat cgatcgagac 1620 gttggccagctatcaaacag ataagaacta cccaaatatt tcctaaatcc agaacggaag 1680 acccattgactaggtcctta cctctcaaat agacagacta ttcttctcca catcaaaata 1740 tagggactcccgatgcaaca aacacgggcc accacacaac aatggtgaaa tgaccatgca 1800 tgcatccacgtccgtacgca gccatttcgt ctataaattt gcttcccatc cgattcaact 1860 acaagcttgcgggcaaaaat ggcaaaggct c 1891

What is claimed is:
 1. A method of producing hybrid seed comprisingincorporating a first expression cassette into a first plant to generatea hemizygous female parent plant, said first expression cassettecomprising: (a) a first gene promoter sequence which is a male flowerspecific promoter; (b) a disrupter gene encoding a protein capable ofdisrupting male fertility operably linked to the first gene promotersequence; (c) a second gene promoter sequence which is a female tissuespecific promoter sequence optionally operably linked to one or moretranslational enhancer or intron sequences; (d) a restorer gene encodinga protein capable of restoring female fertility operably linked to thesecond gene promoter sequence; (e) a third gene promoter sequenceresponsive to the presence or absence of an exogenous chemical inducersaid third gene promoter sequence optionally operably linked to one ormore translational enhancer or intron sequences; and (f) a restorer geneencoding a protein capable of restoring male fertility operably linkedto the third gene promoter sequence; whereby the presence of theexogenous chemical inducer controls male fertility; and incorporating asecond expression cassette into a second plant to generate a hemizygousmale parent plant, said second expression cassette comprising: (g) afirst gene promoter sequence which is a female flower specific promotersequence; (h) a disrupter gene encoding a protein capable of disruptingfemale fertility; (i) a second gene promoter sequence which is a maletissue specific promoter sequence optionally operably linked to one ormore enhancer or intron sequences; (j) a restorer gene encoding aprotein capable of restoring male fertility operably linked to thesecond gene promoter sequence; (k) a third gene promoter sequenceresponsive to the presence or absence of an exogenous chemical inducer,said third gene promoter sequence optionally operably linked to one ormore enhancer or intron sequences; and (l) a restorer gene encoding aprotein capable of restoring female fertility operably linked to thethird gene promoter sequence; whereby the presence of the exogenouschemical inducer controls female fertility; applying an exogenouschemical inducer to the transformants thereby allowing the plants toself-pollinate; growing up plants from the resulting seed; selecting formale and female homozygous plants; crossing the selected male and femaleplants; and obtaining the resulting hybrid seed.
 2. A method accordingto claim 1 wherein the male flower specific promoter sequence is agametophytic promoter sequence.
 3. A method according to claim 1 whereinthe male flower specific promoter sequence is a sporophytic promotersequence.
 4. A method according to claim 1 wherein the plants areselected from the group consisting of wheat, rice, maize, tomato,sunflower, sugarbeet, canola, cotton, soybean and other vegetables.
 5. Amethod according to claim 1 wherein the disrupter gene encoding aproduct capable of disrupting male fertility encodes a product which iscapable of disrupting pollen production.
 6. A method according to claim1 wherein the third gene promoter sequece is the AlcA promoter sequenceor the GST-27 promoter sequence.
 7. A method according to claim 1wherein the restorer gene encoding a product capable of restoring malefertility encodes a product which is capable of restoring pollenproducting.
 8. Hybrid seed resulting from the method of claim 1, whichhybrid seed comprises the first and second expression cassettesaccording to claim 1.